Cytochrome P-450 (hereinafter referred to as "P-450") is a hemoprotein existing in various kinds of living organisms widely from microorganisms to mammals. It has been known that, in order for P-450 to be functional, it is essential to contain a heme in its molecule.
In mammalian adrenals, several types of P-450 species exist, regulating the biosynthesis of steroid hormones. P-450.sub.17.alpha. is a member of the P-450 family found in microsomes in the adrenal cortex of mammals, and has 17-hydroxylase activity towards pregnenolone and progesterone, as well as C17-20-lyase activity towards 17hydroxypregnenolone and 17-hydroxyprogesterone.
Zuber et al. have elucidated the complete DNA sequence of the cDNA encoding the bovine adrenocortical cytochrome P-450.sub.17.alpha. (hereinafter referred to as P-450.sub.17.alpha.) [J. Biol, Chem. 261, 2475 (1986)]and introduced the cDNA in COS1 cells which originally have no ability to produce P-450.sub.17.alpha. and succeeded in expression of the cDNA in the COS1 cells [Science, 234, 1258 (1986)]. The COS1 cells hydroxylated pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, and subsequently converted them to dehydroepiandrosterone and androstenedione, respectively.
It has been known that, when using progesterone as a substrate, for example P-450.sub.17.alpha. exhibits the 17.alpha.-hydroxylase activity to form 17-hydroxyprogesterone, and the C.sub.17-20 -lyase activity to subsequently convert 17-hydroxyprogesterone into androstenedione. The P-450.sub.17.alpha. produced from the said COS1 cells exhibits both of the 17.alpha. hydroxylase activity and C.sub.17-20 -lyase activity. The C.sub.17-20 -lyase activity of the P-450.sub.17.alpha. is about 5% of the 17.alpha.-hydroxylase activity of the P-450.sub.17.alpha..
For the production of cortisol through the following production route using P-450.sub.17.alpha. as a hydroxylating agent, the C.sub.17-20 -lyase activity of the P-450.sub.17.alpha. is considered undesirable because it causes side reactions and by-products: ##STR1##
Accordingly, as a hydroxylating agent, the P-450.sub.17.alpha. with no or least C.sub.17-20 -lyase activity as against the 17-hydroxylase activity is desirable, because the production of by-products can be avoided. From this viewpoint, the said COS1 cells have a defect as a steroid hydroxylating agent.
The present inventors have previously succeeded in the expression of rat cytochrome P-450MC cDNA in Saccharomyces cerevisiae at high expression levels using yeast alcohol dehydrogenase gene promoter (ADH promoter). However, the same cDNA could not be expressed at all when it was connected to GAL10 promoter. The present inventors have also constructed yeast expression plasmids by inserting the DNA sequence coding for bovine adrenal cytochrome P-450scc, which has a side-chain cleaving activity against cholesterol, in an expression plasmid containing the ADH promoter and transformed Saccharomyces cerevisiae with the resulting plasmids, but the transformant yeasts only produced inactive P-450scc with no heme in its molecule. Thus, it was found that it was very difficult to forecast whether or not a particular mammalian P-450 species was able to successfully be expressed in given circumstances, and that each mammalian P-450 species differed from others in terms of its expressible conditions.
The present inventors have studied on the hydroxylation of steroids with P-450.sub.17.alpha. and, as a result of the study, have found that the yeasts which are transformed with the plasmids constructed by inserting the cDNA coding for the bovine adrenocortical cytochrome P-450.sub.17.alpha. in a yeast expression plasmid having ADH promoter produce active P-450.sub.17.alpha. containing a heme in its molecule, and that the P-450.sub.17.alpha. produced by said yeasts is very active in 17-hydroxylase activity, but almost inactive in C.sub.17-20 -lyase activity. The yeasts producing the P-450.sub.17.alpha. quantitatively converted progesterone to 17-hydroxyprogesterone but did not cleave the carbon side chain at 17-20 positions. It has now become possible to selectively hydroxylate steroids with the P-450.sub.17.alpha. -producing-yeasts of the present invention.
The yeasts of the present invention have various advantages over the animal cells of Zuber et al. as a hydroxylating agent for steroids in that, for example, they can easily be grown at a higher cell density (the highest cell density; yeasts: 10.sup.9 cells/ml; animal cells: 10.sup.6 cells/ml) and in a shorter period of time (doubling time; yeasts: 2-3 hours; animal cells: ca. 70 hours) and therefore the hydroxylation of steroids can be performed within a shorter period of time with a smaller vessel with the transformed yeasts of the invention.